Techniques for the injection of DNA and mRNA into mammalian tissue for the purposes of immunization against an expression product have been previously described. See, e.g., European Patent Specification EP 0 500 799 and U.S. Pat. No. 5,589,466. The techniques, termed “nucleic acid immunization” herein, have been shown to elicit both humoral and cell-mediated immune responses. For example, sera from mice immunized with a DNA construct encoding the envelope glycoprotein, gp160, were shown to react with recombinant gp160 in immunoassays, and lymphocytes from the injected mice were shown to proliferate in response to recombinant gp120. Wang et al. (1993) Proc. Natl. Acad. Sci. USA 90:4156–4160. Similarly, mice immunized with a human growth hormone (hGH) gene demonstrated an antibody-based immune response. Tang et al. (1992) Nature 356:152–154. Intramuscular injection of DNA encoding influenza nucleoprotein driven by a mammalian promoter has been shown to elicit a CD8+ cytolytic T lymphocyte (CTL) response that can protect mice against subsequent lethal challenge with virus. Ulmer et al. (1993) Science 259:1745–1749. Immunohistochemical studies of the injection site revealed that the DNA was taken up by myeloblasts, and cytoplasmic production of viral protein could be demonstrated for at least 6 months.
These so-called “genetic vaccines” have thus been demonstrated to elicit immune response in treated animals similar to those observed following administration of live attenuated vaccines, the most effective form of vaccine in use today. The theoretical effectiveness of nucleic acid-based vaccines stems from the ability of the vaccine compositions to elicit the de novo production of correctly folded protein antigens, which can result in the elicitation of antibody responses recognizing complex three dimensional epitopes. In addition, the in vivo production of these antigens in professional antigen presenting cells results in the presentation of processed peptide fragments by MHC class I molecules, resulting in the activation and participation of antigen-specific cytolytic T lymphocytes (CTLs).
To date, the most effective nucleic acid immunization technique involves delivery of a DNA vaccine composition via particle-mediated, intracellular delivery into the epidermis. See, e.g., European Patent No. 0 500 799. This technique avoids the pitfalls of extracellular delivery (e.g., via needle and syringe delivery to skin or muscle) since it is believed that most of such extracellularly delivered DNA is rapidly degraded, necessitating that an excess amount of DNA be inoculated just to achieve a sufficient level of antigen expression. Particle-mediated DNA delivery to the epidermis, on the other hand, achieves the direct, intracellular deposition of plasmid DNAs into epidermal cells, including epidermal Langerhans cells. Because of the direct intracellular delivery, the DNA is protected from extracellular nucleases and only very small quantities of DNA need be delivered. In fact, particle-mediated immunization with nanogram quantities of a given plasmid DNA can result in the elicitation of very strong humoral and CTL responses, often following a single administration.
Malaria is a widespread and significant human disease, particularly in tropical countries. Disease is caused by the mosquito borne parasites Plasmodium falciparum and Plasmodium vivax. Yet malaria vaccine research has yet to result in the development of a safe, practical, and effective prophylactic vaccine product. Although numerous examples of significant immune response have been elicited in both animal models and human volunteers using a variety of experimental vaccines, the actual protection afforded by such vaccinations in human clinical trials has been disappointingly low. The increased scientific understanding of the life cycle of the malaria parasite and the identification of important antigens from various parasite life stages have allowed the development of a variety of vaccine strategies designed to elicit the formation of protective antibodies as well as cellular effector cells such as CTLs.
The disease cycle of malaria begins with a mosquito bite that injects the infectious malaria sporozoites into the bloodstream. It would thus seem reasonable that sporozoite-specific antibodies would be an effective means of preventing or significantly limiting the infection of hepatocytes at this stage. Following hepatocyte infection, however, the parasite develops intracellularly in the hepatocyte and may escape circulating antibodies. At this stage, the presentation of sporozoite-specific antigens on the surface of the infected hepatocytes prior to merozoite or blood stage merozoite release provides an attractive target for malaria-specific CTLs. Such effector cells could either eliminate the infected hepatocytes or elicit the destruction of intracellular parasites, prior to first release of mature blood stage merozoites.